Autophagy Plays A Protective Role In Advanced Glycation End Product-induced Apoptosis In Human Periodontal Ligament Cells

Periodontitis is a common,chronic inflammatory disease of the supporting structures of the teeth.It is highly prevalent and is the most important cause of tooth loss in adults.The pathogenesis of the disease is complicated,in addition to pathogenic microorganisms in the biofilm,genetic and environmental factors,especially systemic diseases,contribute to the cause of it.Diabetes mellitus（DM）is a group of chronic metabolic disorders characterized by abnormal glucose metabolism.This condition constitutes a serious public health concern:it requires lifelong care,leads to premature death and remains incurable.The association between the two diseases has been described as bidirectional.Periodontitis negatively affects glycemic control.The incidence and severity of periodontitis in patients with DM higher than in individuals without DM.Periodontitis has been regarded as the sixth major complication of DM,but the underlying pathogenesis of their correlations is not yet clear.Advanced glycation end products（AGEs）are stable covalent compounds produced by the non-enzymatic reaction（called the"Maillard reaction"）of glucose and proteins,lipids and nucleic acids.Numerous studies have shown that AGEs play an important role in the development of diabetic complications and many inflammatory reactions.However,those studies still could not fully elucidate the pathways or potential effects of AGEs on the periodontitis in patients with DM.In our study,the aim was to survey the potential mechanism of apoptosis and autophagy flux in AGEs-treated human periodontal ligament cells（HPDLCs）via an in vitro model,and to further explore the significant influence of AGEs on periodontitis in DM patients.It will help to select appropriate intervention targets and provide new ideas for the prevention and treatment of periodontitis in DM patients.Part one:AGEs induced apoptosis by the mitochondrial apoptosis pathway in HPDLCsObjective:To investigate the apoptosis of HPDLCs induced by AGEs and its potential molecular mechanism via an in vitro model.Methods:Periodontal ligament tissues were isolated from the extracted premolar teeth due to orthodontic treatment.Cultured HPDLCs were treated with different concentrations（0,50,100,200,250,and 300μg/m L）of AGEs for 0,1,3,6,12,and24 h,and BSA was used as a control.The CCK-8 assay was used to assess the cell viability,the degree of apoptosis was evaluated in the cell death detection ELISA^PLUSkit and the percentage of apoptotic cells were determined by flow cytometry.HPDLCs were treated with 200μg/m L AGEs for 0,0.5,1,3 and 6 h,Intracellular ROS levels were analyzed using the fluorescent probe DCFH-DA and the mitochondrial membrane potential（MMP）were measured using the fluorescent dye JC-1.HPDLCs were treated with 200μg/m L AGEs for 12h,the molecules related with apoptosis were analyzed by Western blotting analysisResults:Compared with the control group,the treatment of AGEs（200μg/m L）for12 h in the HPDLCs significantly decreased the survival rate of cells,and increased the DNA fragmentation ratio and apoptotic ratio of the cells.Z-VAD-fmk（a pan-caspase inhibitor）,was pre-treated with cells significantly decreased the ratio of apoptotic cells（P<0.001）.ROS production increased as early as 0.5 h in HPDLCs treated with AGEs（P<0.01）.The accumulation of JC-1 monomers in the AGEs-treated cells significantly increased at 1 h（P<0.01）.However,a reduced formation of JC-1 monomers was shown after pre-treatment with a ROS scavenger NAC（P<0.01）.The expression of Bax,cleaved Caspase-3 and cleaved PARP were significantly increased after the treatment of AGEs for 12h,however,the expression of Bcl-2,as antiapoptotic proteins,was decreased compared with the control group（P<0.01）.In HPDLCs pre-treated with NAC（5m M）,the levels of these molecules was reversed significantly（P<0.01）.Conclusion:AGEs can induce the depolarization of MMP after the production of ROS,lead to the mitochondrial dysfunction and participate in the mitochondrial apoptosis pathway in HPDLCs.Part two:AGEs triggered autophagy flux via ROS-mediated ERK signaling pathway in HPDLCsObjective:To investigate the effect of AGEs on autophagy flux in HPDLCs and the role of ROS/ERK signaling pathway in autophagy via an in vitro model.Methods:HPDLCs were treated with 200μg/m L AGEs for 0,1,3,6,12 and 24 h,or incubated with 200μg/m L AGEs for 3 h with or without the pre-treatment of 3-MA and RAPA.The LC3-II/LC3-I ratio and p62 were detected by Western blotting.Autophagosomes were observed by transmission electron microscopy.Cells were incubated with AGEs（200μg/ml）for 3 h and 12h.Fluorescence microscopy was used to show the yellow puncta（autophagosomes）and red puncta（autolysosomes）in HPDLCs.Cells were incubated with 200μg/m L AGEs for 3 h with or without the pre-treatment of U0126,NAC and the small interfering RNA（si RNA）against ERK1/2（si ERK1/2）.Western blotting was used to show the levels of MEK1/2,ERK1/2 and LC3-II/LC3-I in HPDLCs.Results:Compared with the control group,the ratio of LC3II/LC3I and the formation of autophagosomes were significantly increased in HPDLCs incubated with 200μg/m L AGEs for 3 h（P<0.01）.A serious decrease in the LC3-II/LC3-I expression with 3-MA and a further increase in the LC3-II expression with RAPA（P<0.01）.The increased expression of p-MEK1/2,p-ERK1/2,and LC3II/LC3I ratio and decreased expression of p62 were significantly in AGEs-treated HPDLCs for 3 h（P<0.01）.Additionally,pretreatment with U0126,NAC or si ERK1/2,the levels of the ERK signalling pathway related proteins and LC3II/LC3I conversion were decreased and the p62 expression was increased（P<0.01）.Conclusion:AGEs can induce autophagic flux in HPDLCs through ROS/ERK signalling pathway.Part three:Autophagy plays a protective role in AGEs-induced apoptosis of HPDLCsObjective:To investigate the role of autophagy in AGEs-induced apoptosis of HPDLCs.Methods:The HPDLCs were exposed to AGEs with or without the pretreatment of U0126,3-MA or RAPA for 3h,the cell viability was assessed by CCK-8 assay,the percentage of apoptotic cells was detected by flow cytometry and the apoptosis-related protein Bcl-2,Bax,Caspase-3 and PARP levels were shown by western blotting.Results:Compared with the AGEs group,pre-treatments with U0126 and 3-MA markedly decreased the cell viability and increased the ratio of apoptotic cells induced by AGEs（P<0.01）,as well as pre-treatments with RAPA,triggered the opposite effects（P<0.01）.In addition,U0126 could further increase the BAX,cleaved PARP-1 and cleaved caspase-3 levels and reduced Bcl-2 levels（P<0.01）.However,RAPA triggered the opposite effect of the apoptosis-related molecules（P<0.01）.Conclusion:The inhibition of autophagy flux can aggravate AGEs-induced apoptosis,in the meanwhile,the inducer of autophagy declines the apoptosis.Therefore,the autophagy has a protective influence on apoptosis induced by AGEs in HPDLCs.